User:Karmella Haynes/Notebook/BioBrick cloning/2015/06/24

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06/24/15

  • Cas-tone - H3#_MV11 minipreps, digests



Cas-tone - H3#_MV11 minipreps, digests

  • Cultures
    • Pick 2 colonies from each ligation plate
    • Make streak library & 5 mL cultures
  • Digests
    • Sigma GenElute kit; elute w/ 75μL eln. buffer
    • Check with S/HindIII digests - H3 insertion destroys SpeI site
    • Note: Fragment without H3 part = 7162; added H3-1-# size + 7162 to get lengths for longer H/H band
    • H3-1-40 = 129; H3-1-60 = 189; H3-1-80 = 250; H3-1-116 = 373; H3-1-136 = 433
Reagent Volume Expected:
1,2. H3-1-40_MV11 = 2345, 7291
3,4. H3-1-60_MV11 = 2345, 7351
5,6. H3-1-80_MV11 = 2345, 7412
7,8. H3-1-116_MV11 = 2345, 7535
9,10. H3-1-136_MV11 = 2345, 7595
Empty MV11 = 6131, 2345, 1028
Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
SpeI 1.0
HindIII 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~15 min.


  • DNA concentrations: BioTek Synergy H1 Take 3
Sample OD260 260/280 ng/μL
1. H340_MV11 a 0.194 1.885 194.084
2. H340_MV11 b 0.154 1.936 153.756
3. H360_MV11 a 0.199 1.903 199.259
4. H360_MV11 b 0.135 1.913 135.024
5. H380_MV11 a 0.179 1.931 178.795
6. H380_MV11 b 0.057 2.000 56.959
7. H3116_MV11 a 0.148 1.883 148.401
8. H3116_MV11 b 0.091 1.951 91.09
9. H3136_MV11 a 0.145 1.884 145.389
10. H3136_MV11 b 0.151 1.898 150.727


CONCLUSION

  • Success! Clones 1-5, 7-10 look correct. Clone 6 (H380_MV11 b) is empty vector
  • Verify via sequencing
  • Sequencing order (6/25/15) - #10265
  1. H340-MV11-a, CMVf
  2. H340-MV11-a, DD123
  3. H340-MV11-b, CMVf
  4. H340-MV11-b, DD123
  5. H360-MV11-a, CMVf
  6. H360-MV11-a, DD123
  7. H360-MV11-b, CMVf
  8. H360-MV11-b, DD123
  9. H380-MV11-a, CMVf
  10. H380-MV11-a, DD123
  11. H3116-MV11-a, CMVf
  12. H3116-MV11-a, DD123
  13. H3116-MV11-b, CMVf
  14. H3116-MV11-b, DD123
  15. H3136-MV11-a, CMVf
  16. H3136-MV11-a, DD123
  17. H3136-MV11-b, CMVf
  18. H3136-MV11-b, DD123